Project I. piRNA function in epigenetic inheritance. Craig Mello, P.I. Project summary Darwin recognized that evolution by natural selection required two self-evident but entirely mysterious biological mechanisms: (i) a property of inheritance such that offspring share features of parents, and (ii) a source of variation so that novel inherited features, adaptations, can arise. The discovery of Mendelian genetics and of the structure and properties of DNA seemed to solve these mysteries, creating the widely held DNA-centric view of inheritance. However, beginning with Barbara McClintock's classic work on variegation in maize, a growing field of epigenetics has begun to reveal how RNA and chromatin can contribute to inheritance. Recently, we have shown that an RNAi-related small-RNA pathway called the Piwi pathway can initiate a very stable transgenerational mode of epigenetic silencing in the C. elegans germline. Remarkably, this pathway involves piRNA signals that constitute not only a memory (and enforcer) of gene silencing but also involves an epigenetic memory (and protector) of expressed germline genes. PIWI Argonautes and their small RNA cofactors (piRNAs) are conserved regulators of germline development in animals. piRNA-pathway mutants exhibit embryonic lethality and partial or complete deficits in fertility, characterized by under- proliferated germlines and activation of transposable elements. While the role of piRNAs in transposon silencing is well established, how piRNAs promote germ cell development and function is not understood. Here we propose a set of genetic, biochemical and bioinformatic studies to further characterize and understand: 1) the molecular mechanisms and outcomes of targeting by PIWI/piRNA complexes, 2) the specificity of individual PIWIs, and 3) the epigenetic functions of piRNA pathways. Insights from parallel projects in the laboratories of Drs. Theurkauf and Zamore, with informatics-based model building by the Weng lab, will inform this analysis and provide new tools and methods for probing piRNA activities and downstream functions. Relevance These studies will advance our basic understanding of piRNA function in fertility and epigenetic inheritance, and should guide our understanding of how related pathways protect the human genome, maintain somatic cells, and promote the pluripotency and immortality of germ cells.